Evaluation of a Novel Non-Penetrating Electrode for Use in DNA Vaccination

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use with a wide variety of diseases. Alone, this technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. In this study we evaluated the MEA for its use in DNA vaccination using Hepatitis B virus as the infectious model. We utilized the guinea pig model because their skin is similar in thickness and morphology to humans. The plasmid encoding Hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to injection only groups. While this electrode requires further study, our results suggest that the MEA has potential for use in electrically mediated intradermal DNA vaccination

Electro-Gene Transfer to Skin Using a Noninvasive Multielectrode Array

Because of its large surface area and easy access for both delivery and monitoring, the skin is an attractive target for gene therapy for cutaneous diseases, vaccinations and several metabolic disorders. The critical factors for DNA delivery to the skin by electroporation (EP) are effective expression levels and minimal or no tissue damage. Here, we evaluated the non-invasive multielectrode array (MEA) for gene electrotransfer. For these studies we utilized a guinea pig model, which has been shown to have a similar thickness and structure to human skin. Our results demonstrate significantly increased gene expression 2 to 3 logs above injection of plasmid DNA alone over 15 days. Furthermore, gene expression could be enhanced by increasing the size of the treatment area. Transgene-expressing cells were observed exclusively in the epidermal layer of the skin. In contrast to caliper or plate electrodes, skin EP with the MEA greatly reduced muscle twitching and resulted in minimal and completely recoverable skin damage. These results suggest that EP with MEA can be an efficient and non-invasive skin delivery method with less adverse side effects than other EP delivery systems and promising clinical applications

Increased Tissue Temperature Improves Electro-Transfer Mediated Gene Delivery to Skin

[Introduction] Developing an optimal gene electro-transfer system for delivery of plasmid DNA to the skin in vivo has been challenging with expression often confined in the epithelium requiring high voltage electric fields, which can cause cellular and tissue damage. Minimizing such damage, while optimizing gene expression profiles, are highly desirable for therapeutic applications of gene delivery to the skin. Cell membrane fluidity is temperature dependent, thus moderate temperature elevation can increase membrane fluidity. Utilizing this concept, we developed a novel gene electro-transfer electrode for in vivo applications with ability to heat tissue with a laser integrated into a four pin multi-electrode array, to improve gene delivery efficiency

Evaluation of Delivery Conditions for Cutaneous Plasmid Electrotransfer Using a Multielectrode Array

Electroporation (EP) is a simple in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of tissues. Delivery of plasmid DNA by EP to a large surface area is not practical because the distance between the electrode pairs, and therefore the applied voltage, must be increased to effectively permeabilize the cell membrane. The design of the multielectrode array (MEA) incorporates multiple electrode pairs at a fixed distance to allow for delivery of plasmid DNA to the skin, potentially reducing the sensation associated with in vivo EP. In this report, we evaluate the effects of field strength and pulse width on transgene expression and duration using a plasmid encoding the luciferase reporter gene delivered by intradermal injection in a guinea pig model followed by EP with the MEA. As expected, the level of luciferase expression increased with the magnitude and duration of the voltage applied. In addition to adjusting transgene expression levels by altering fielding strength, levels could also be controlled by adjusting the plasmid dose. Our results indicate that the design of the MEA is a viable option for cutaneous plasmid DNA delivery by in vivo EP to a large surface area

Thermal Assisted In Vivo Gene Electrotransfer

Gene electrotransfer is an effective approach for delivering plasmid DNA to a variety of tissues. Delivery of molecules with electric pulses requires control of the electrical parameters to achieve effective delivery. Since discomfort or tissue damage may occur with high applied voltage, the reduction of the applied voltage while achieving the desired expression may be an important improvement. One possible approach is to combine electrotransfer with exogenously applied heat. Previous work performed in vitro demonstrated that increasing temperature before pulsing can enhance gene expression and made it possible to reduce electric fields while maintaining expression levels. In the study reported here, this combination was evaluated in vivo using a novel electrode device designed with an inserted laser for application of heat. The results obtained in this study demonstrated that increased temperature during electrotransfer increased expression or maintained expression with a reduction in applied voltage. With further optimization this approach may provide the basis for both a novel method and a novel instrument that may greatly enhance translation of gene electrotransfer

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after geneelectrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest geneelectrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient

Development of a Non-Invasive Electrode for Intradermal Electrically Mediated DNA Vaccination

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. This study evaluated the use of the MEA for the development of DNA vaccines. We assessed the appropriate delivery conditions for gene expression and the development of humoral immunity. We used both B. anthracis and HBV as infectious models for our experiments. Our results indicated that the MEA can enhance gene expression in a mouse model with minimal to no tissue damage. Optimal delivery conditions, based on generation of antibodies, were determined to be 125-175V/cm and 150ms with 200ug and a prime boost protocol administered on Day 0 and 14. Under these conditions, end-point titers of 20,000-25,000 were generated. Neutralizing antibodies were noted in 40-60% of animals. Additionally, we utilized a guinea pig model to assess the translation potential of this electrode. The plasmid encoding HBsAg, pHBsAg, was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in the experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to the injection only groups. Results from both experimental models demonstrate that protective levels of humoral immunity can be generated and that this electrode should translate well to the clinic

Topical gene electrotransfer to the epidermis of hairless guinea pig by non-invasive multielectrode array.

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient